Structural Requirements for Efficient Processing and Activation of Recombinant Human UDP-N-acetylglucosamine:Lysosomal-enzyme-N-acetylglucosamine-1-phosphotransferase

Abstract

Mannose 6-phosphate-modified N-glycans are the determinant for intracellular targeting of newly synthesized lysosomal hydrolases to the lysosome. The enzyme responsible for the initial step in the synthesis of mannose 6-phosphate is UDP-N-acetylglucosamine:lysosomalenzyme-N-acetylglucosmine-1-phosphotransferase(GlcNAc-phosphotransferase). GlcNAc-phosphotransferase is a multisubunit enzyme with an ␣ 2 ␤ 2 ␥ 2 arrangement that requires a detergent for solubilization. Recent cloning of cDNAs and genes encoding these subunits revealed that the ␣-and ␤-subunits are encoded by a single gene as a precursor, whereas the ␥-subunit is encoded by a second gene. The hydropathy plots of the deduced amino acid sequences suggested that the ␣-and ␤-subunits but not the ␥-subunit contain transmembrane domains. Access to these cDNAs allowed us to express a soluble form of human recombinant GlcNAc-phosphotransferase by removing the putative transmembrane and cytoplasmic domains from the ␣-and ␤-subunits. Because this modification prevented precursor processing to mature ␣-and ␤-subunits, the native cleavage sequence was replaced by a cleavage site for furin. When the modified ␣/␤-subunits (␣/␤-subunits) precursor and wild type ␥-subunit cDNAs were co-expressed in 293T or CHO-K1 cells, a furin-like protease activity in these cells cleaved the precursor and produced an active and processed soluble GlcNAc-phosphotransferase with an ␣ 2 ␤ 2 ␥ 2 -subunits arrangement. Recombinant soluble GlcNAc-phosphotransferase exhibited specific activity and substrate preferences similar to the wild type bovine GlcNAcphosphotransferase and was able to phosphorylate a lysosomal hydrolase, acid ␣-glucosidase in vitro.Most lysosomal hydrolases are targeted to the lysosome via mannose 6-phosphate (Man-6-P) 2 -modified N-glycans. The initial step of Man-6-P biosynthesis is catalyzed by the membrane-bound enzyme UDP-N-acetylglucosamine:lysosomal-enzyme-N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase, EC 2.7.8.17), which transfers GlcNAc 1-phosphate from UDP-GlcNAc to mannose in high mannose-type glycans (1-3). The second step is removal of GlcNAc by N-acetylglucosamine-1-phosphodiester ␣-GlcNAcase (UCE) to expose mannose 6-phosphate (4, 5).Previously, we reported that the bovine GlcNAc-phosphotransferase has an ␣ 2 ␤ 2 ␥ 2 -configuration (6). We also found that GlcNAc-phosphotransferase is encoded by two genes; one encoding the ␣/␤-subunits precursor (7,8) and the other, the ␥-subunit (9). Characterization of the cDNAs revealed that the ␣-and ␤-subunits contain amino and carboxyl terminus transmembrane domains, respectively, whereas the ␥-subunit does not. The mechanism responsible for the processing of the ␣/␤-subunits precursor into the mature ␣-and ␤-subunits is not yet known, but likely to be the result of protease(s) in the endoplasmic reticulum (ER) or cis-Golgi, where GlcNAc-phosphotransferase resides (24). Access to the cDNAs for all three subunits of GlcNAc-phosphotransferase made it possible to express recombinant GlcNAc-phos...

Mucolipidosis II and III alpha/beta: mutation analysis of 40 Japanese patients showed genotype–phenotype correlation

“…The above two mutations consequently lead to frameshift. The Korean group reported skipping of exon 13 only in ML III g (1) g (6) g (6) g (2) g 4 3…”

“…The a-b boundary is located within exon 14, but the detailed mechanism of processing the precursor into a and b subunits is not clear. 6 A series of genetic-complementation studies have shown heterogeneity in ML III and the genetic relationship between ML II and III. [7][8][9] Mutations in GNPTAB cause both the severe type of ML (ML II alpha/beta, ML II, I-cell disease (MIM 252500)) and the attenuated type of ML (ML III alpha/beta, ML IIIA, PseudoHurler polydystrophy (MIM 252600)).…”

Multiple Domains of GlcNAc-1-phosphotransferase Mediate Recognition of Lysosomal Enzymes

“…The GNPTAB gene encodes the ␣ and ␤ subunits, whereas the GNPTG gene encodes the ␥ subunit. Enzyme kinetic studies have indicated that the ␣ and ␤ subunits specifically bind lysosomal acid hydrolases and mediate the catalytic function of the enzyme (8,9). The ␥ subunit enhances the rate of GlcNAc-P transfer to a subset of the acid hydrolases without substantially altering the binding to these acceptors.…”

“…The ␣/␤ subunits contain three identifiable domains as follows: the Stealth domain, two Notch repeat modules, and a DNA methyltransferase-associated protein (DMAP) interaction domain. The Stealth domain resembles sequences within bacterial genes that encode sugar-phosphate transferases involved in cell wall polysaccharide synthesis and has been shown to mediate the catalytic function of GlcNAc-1-phosphotransferase (8,10,11). Importantly, the bacterial genes lack the Notch repeats and the DMAP interaction domain, which has been proposed to function as a protein-protein interaction domain (12).…”

Production of α-L-iduronidase in maize for the potential treatment of a human lysosomal storage disease

“…Most lysosomal enzymes including αliduronidase require an M6P tag for efficient uptake/lysosomal delivery in human cells. We investigated whether the phosphorylation of Nglycan terminal mannose residues of the 'test' αliduronidase could be achieved in vitro using recombinant soluble UDP-GlcNAc:lysosomal enzyme Nacetylglucosamine1phosphotransferase 21 (Fig. 6b).…”